[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis (IM) and is associated with epithelial cell malignancies such as nasopharyngeal carcinoma and gastric carcinoma, as well as lymphoid malignancies including Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma and post-transplant lymphoproliferative disorder. EBV vaccines to prevent primary infection or disease, or therapeutic vaccines to treat EBV malignancies have not been licensed. Most efforts to develop prophylactic vaccines have focused on EBV gp350, which is the major target of neutralizing antibody. A single phase 2 trial of an EBV gp350 vaccine has been reported; the vaccine reduced the rate of IM but not virus infection. The observation that infusion of EBV-specific T cells can reduce disease due to Hodgkin lymphoma and nasopharyngeal carcinoma provides a proof of principle that a therapeutic vaccine for these and other EBV-associated malignancies might be effective. Most therapeutic vaccines have targeted EBV LMP2 and EBV nuclear antigen-1. As EBV is associated with nearly 200 000 new malignancies each year worldwide, an EBV vaccine to prevent these diseases is needed.
[Show abstract][Hide abstract] ABSTRACT: No Herpes simplex virus type-2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular injection of HSV-2 gB and gD proteins in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) and gD (gDsec), but not PsV expressing cytoplasmic or membrane-bound forms, induced circulating and intravaginal tissue resident memory CD8(+) T cells able to secrete IFN-γ and TNF-α and moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec/gDsec vaccines conferred improved survival after HSV-2 vaginal challenge compared to HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with reduced severity of genital lesions and lower viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with soluble HSV-2 gD in alum and monophosphoryl lipid A (gD2t/Alum/MPL) elicited high neutralizing antibody titers and improved survival, but did not reduce genital lesions and viral shedding. Vaccination combining HPV-gBsec/gDsec ivag and gD2t/Alum/MPL i.m. improved survival and reduced genital lesions and viral shedding. Finally, circulating HSV-2-specific CD8(+) T cells, not serum antibodies, correlated with reduced viral shedding. Together our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.
[Show abstract][Hide abstract] ABSTRACT: A high-throughput test to detect varicella-zoster virus (VZV) antibody in varicella vaccine recipients is not available. One of the most sensitive tests to detect VZV antibodies after vaccination is the fluorescent antibody to membrane antigen test (FAMA). Unfortunately, this test is labor intensive, somewhat subjective to read, and not commercially available. Therefore, we developed a highly quantitative and high-throughput luciferase immunoprecipitation systems (LIPS) assay to detect antibody to VZV glycoprotein E (gE). Testing of children who received the varicella vaccine showed that the gE LIPS assay had 90% sensitivity and 70% specificity, a viral capsid antigen ELISA assay had 67% and 87% specificity, and a glycoprotein ELISA (not commercially available in the US) had 94% sensitivity and 74% specificity, compared with the FAMA test. Rates of antibody detection by the gE LIPS and glycoprotein ELISA were not statistically different. Therefore, the gE LIPS assay may be useful for detecting VZV antibody in varicella vaccine recipients.
Clinical and vaccine Immunology: CVI 07/2014; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) infection can cause severe disease in neonates and immunocompromised persons, and infectious mononucleosis in healthy adults. While, rhesus CMV (RhCMV) infects human cells in culture, it is unknown whether the virus can infect humans.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 05/2014; · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herpesvirus capsid morphogenesis occurs in the nucleus, while final maturation takes place in the cytosol requiring translocation of capsids through the nuclear envelope. The nuclear egress complex consisting of homologs of herpes simplex virus pUL31 and pUL34, is required for efficient nuclear egress via primary envelopment-deenvelopment. Recently, we described an alternative mode of nuclear escape by fragmentation of the nuclear envelope induced by replication competent pUL31 and pUL34 deletion mutants of the alphaherpesvirus pseudorabies virus (PrV), which had been selected by serial passaging in cell culture. Both passaged viruses carry congruent mutations in seven genes including UL46 which encodes one of the major tegument proteins. Herpesvirus pUL46 homologs have recently been shown to activate the PI3K-Akt and ERK1/2 signaling pathways, which are involved in regulation of mitosis and apoptosis. Since in uninfected cells fragmentation of the nuclear envelope occurs during mitosis and apoptosis, we analyzed whether pUL46 of PrV is involved in signaling events impairing the integrity of the nuclear envelope. We show here that PrV pUL46 is able to induce phosphorylation of ERK1/2 and, thus, expression of ERK1/2 target genes, but fails to activate the PI3K-Akt pathway. Deletion of UL46 from PrV-ΔUL34Pass and PrV-ΔUL31Pass, or substitution by wild type UL46 resulted in enhanced nuclear envelope breakdown indicating that the mutations in pUL46 may limit the extent of NEBD. Thus, although pUL46 induces ERK1/2 phosphorylation, controlling the integrity of the nuclear envelope is independent of the ERK1/2 signaling pathway.
Herpesvirus nucleocapsids can leave the nucleus by regulated, vesicle-mediated transport through the nuclear envelope designated as nuclear egress, or by inducing nuclear envelope breakdown (NEBD). The viral proteins involved in NEBD are unknown. We show here that the pseudorabies virus tegument protein pUL46 induces the ERK1/2 signaling pathway and modulates NEBD. However, these two processes are independent and ERK1/2 signaling induced by pUL46 is not involved in herpesvirus-induced NEBD.
[Show abstract][Hide abstract] ABSTRACT: Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.
Virology 03/2014; s 452–453:52–58. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Human cytomegalovirus (HCMV) infection can cause severe disease in neonates and immunocompromised persons, and infectious mononucleosis in healthy adults. While, rhesus CMV (RhCMV) infects human cells in culture, it is unknown whether the virus can infect humans.
We sought to determine whether primate workers, including those with injuries from animals, might be infected asymptomatically with RhCMV.
We developed serologic assays that distinguish RhCMV from HCMV antibodies. We tested two groups of primate workers: those with documented injuries or mucosal splashes associated with rhesus macaques, and those with no documented exposure who worked with these animals.
None of over 200 primate workers, including 119 with injuries or mucosal splashes associated with exposures to macaques, were seropositive for RhCMV.
The frequency of asymptomatic RhCMV infection in persons who work with rhesus macaques was <0.5% (<1/200 primate workers).
[Show abstract][Hide abstract] ABSTRACT: Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons. We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren's syndrome (SjS) to determine if their antibody profiles differed from control subjects. The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005), and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls. The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.
PLoS ONE 12/2013; 8(12):e81635. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rhesus cytomegalovirus (RhCMV) 68-1 is the prototypic strain of RhCMV that has been used for pathogenesis and vaccine development. We determined the complete sequence of the RhCMV 68-1 UL/b' region directly from the original urine from which RhCMV 68-1 was isolated in 1968, and compared it to other RhCMVs. The laboratory passaged RhCMV 68-1 has inversions, deletions, and stop codons in UL/b' that are absent in the original isolate and other low passage RhCMV isolates. Fourteen of the 17 open reading frames (ORFs) in 68-1 UL/b' in the original isolate share >95% amino acid identity with low passage RhCMV. The original isolate retains 6 ORFs that encode α-chemokine-like proteins, including RhUL146 and RhUL146b that share only 92% and 81% amino acid identity, respectively, with a contemporary low passage RhCMV isolate. Identification of the original RhCMV 68-1 UL/b' sequence is important for using RhCMV 68-1 in pathogenesis and vaccine studies.
[Show abstract][Hide abstract] ABSTRACT: Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. VZV induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of pro-apoptotic proteins Bim and BAD, but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduce their pro-apoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim, but not BAD, in VZV-infected cells was dependent on activation of the MEK/ERK pathway. Cells knocked-down for Bim showed delayed VZV plaque formation resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller sized plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.
[Show abstract][Hide abstract] ABSTRACT: The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.
[Show abstract][Hide abstract] ABSTRACT: The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several malignancies including nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and lymphoma after organ or stem cell transplant. A candidate vaccine containing soluble EBV glycoprotein gp350 protected cottontop tamarins from EBV lymphoma after challenge with EBV. In the only phase 2 trial of an EBV vaccine in humans, soluble gp350 in alum and monophosphoryl lipid A adjuvant reduced the rate of infectious mononucleosis in EBV seronegative adults, but did not affect the rate of EBV infection. A peptide vaccine corresponding to EBV latency proteins has been tested in a small number of adults to prevent infectious mononucleosis. Some of the barriers to development of an EBV vaccine include (a) whether viral proteins in addition to gp350 would be more effective for preventing mononucleosis or EBV malignancies, (b) the difficulty of performing clinical trials to prevent EBV associated malignancies in the absence of good surrogate markers for tumor development, and the long period of time between primary EBV infection and development of many EBV tumors, (c) the lack of knowledge of immune correlates for protection against EBV infection and disease, (d) the limitations in animal models to study protection against EBV infection and disease, and (e) the need for additional information on the economic and societal burden of infectious mononucleosis to assess the cost-benefit of a prophylactic vaccine.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the transcription factor GATA2 underlie the syndrome of monocytopenia, B and NK cell lymphopenia associated with opportunistic infections and cancers. In addition, patients suffer from recurrent and severe viral infections. NK cells play a critical role in mediating anti-viral immunity. Human NK cells are thought to mature in a linear fashion, with the CD56(bright) stage preceding terminal maturation to the CD56(dim) stage, considered the most enabled for cytotoxicity. Here we report an NK cell functional defect in GATA2 deficient patients and extend this genetic lesion to what is considered to be the original NK cell deficient patient. In most cases GATA2 deficiency is accompanied by a severe reduction in peripheral blood NK cells and marked functional impairment. The NK cells detected in peripheral blood of some GATA2 deficient patients are exclusively of the CD56(dim) subset, which is recapitulated upon in vitro NK cell differentiation. In vivo IFNα treatment increased NK cell number and partially restored function, but did not correct the paucity of CD56(bright) cells. Thus, GATA2 is required for the maturation of human NK cells and the maintenance of the CD56(bright) pool in the periphery. Defects in GATA2 are a novel cause of profound NK cell dysfunction.
[Show abstract][Hide abstract] ABSTRACT: After HSV infection, some trigeminal ganglion neurons support productive cycle gene expression, while in other neurons the virus establishes a latent infection. We previously demonstrated that HSV-1 and HSV-2 preferentially establish latent infection in A5+ and KH10+ sensory neurons, respectively, and that exchanging the latency-associated transcript (LAT) between HSV-1 and HSV-2 also exchanges the neuronal preference. Since many viral genes besides the LAT are functionally interchangeable between HSV-1 and HSV-2, we co-infected HSV-1 and HSV-2, both in vivo and in vitro, to determine if trans-acting viral factors regulate whether HSV infection follows a productive or latent pattern of gene expression in sensory neurons. The pattern of HSV-1 and HSV-2 latent infection in trigeminal neurons was no different following co-infection than with either virus alone, consistent with the hypothesis that a trans-acting viral factor is not responsible for the different patterns of latent infection of HSV-1 and HSV-2 in A5+ and KH10+ neurons. Since exchanging the LAT regions between the viruses also exchanges neuronal preferences, we infected transgenic mice that constitutively express 2.8 kb of the LAT region with the heterologous viral serotype. Endogenous expression of LAT did not alter the pattern of latent infection after inoculation with the heterologous serotype virus, demonstrating that the LAT region does not act in trans to direct preferential establishment of latency of HSV-1 and HSV-2. Using HSV1-RFP and HSV2-GFP in adult trigeminal ganglion neurons in vitro, we determined that HSV-1 and HSV-2 do not exert trans-acting effects during acute infection to regulate neuron specificity. Although some neurons were productively infected with both HSV-1 and HSV-2, no A5+ or KH10+ neurons were productively infected with both viruses. Thus, trans-acting viral factors do not regulate preferential permissiveness of A5+ and KH10+ neurons for productive HSV infection and preferential establishment of latent infection.
PLoS ONE 12/2012; 7(12):e53281. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HSV infections are prevalent worldwide. A vaccine to prevent genital herpes would have a significant impact on this disease. Several vaccines have shown promise in animal models; however, so far these have not been successful in human clinical studies. Prophylactic HSV vaccines to prevent HSV infection or disease have focused primarily on eliciting antibody responses. Potent antibody responses are needed to result in sufficiently high levels of virus-specific antibody in the genital tract. Therapeutic vaccines that reduce recurrences need to induce potent T-cell responses at the site of infection. With the increasing incidence of HSV-1 genital herpes, an effective herpes vaccine should protect against both HSV-1 and HSV-2. Novel HSV vaccines, such as replication-defective or attenuated viruses, have elicited humoral and cellular immune responses in preclinical studies. These vaccines and others hold promise in future clinical studies.
Expert Review of Vaccines 12/2012; 11(12):1429-40. · 4.22 Impact Factor